HUMASIS MALARIA ANTIGEN TEST HIGH SENSITIVE DIFFERENTIAL DIAGNOSIS OF MALARIA INFECTION

HUMASIS MALARIA ANTIGEN TEST HIGH SENSITIVE DIFFERENTIAL DIAGNOSIS OF MALARIA INFECTION

References 1) World Malaria Report 2010, WHO 2) Rapid diagnostic tests for malaria parasites, Clin. Microbiol. Rev. 2002, 15(1):66-78 3) The role of rapid diagnostic tests in managing malaria, PLoS Medicine, 2009, 6(4):1-2 4) Malaria rapid diagnosis_making it work, WHO regional office for the western pacific, 2003

What is Malaria? Malaria is a mosquito-borne infectious disease of humans caused by eukaryotic protists of the genus Plasmodium. It is widespread in tropical and subtropical regions, including much of Sub-Saharan Africa, Asia and the Americas. Malaria is prevalent in these regions because of the significant amounts of rainfall and consistent high temperatures; warm, consistent temperatures and high humidity, along with stagnant waters in which their larvae mature, provide mosquitoes with the environment needed for continuous breeding. According to WHO in 2010, there were 247 million malaria cases among 3.3 billion people at risk in 2008 from 109 countries resulting in an estimated 881,000 deaths. These deaths were primarily in Africa (91%) and in children under 5 years of age (85%). Four species of Plasmodium can infect and be transmitted by humans. Severe disease is largely caused by Plasmodium falciparum. Malaria caused by Plasmodium vivax, Plasmodium ovale and Plasmodium malariae is generally a milder disease that is rarely fatal. Table 1. Differentiating features of P.falciparum and P.vivax P.falciparum P.vivax Associated with serious complications e.g. cerebral malaria, jaundice, renal failure etc. including high mortality Relatively benign and rarely produces serious complications or death 35% of RBC s involved Less than 1% of RBC s are parasitized Figure 1. Geographical distribution of Malaria infection. Gametocytes persists in the blood for 30~60 days or more Gametocytic stage persists in the peripheral blood for 2 days

Diagnosis of Malaria Microscopy remains the gold standard for detection of malaria parasitemia as it can provide information on both the species of parasite and parasite density of infection. However, the procedure is labor-intensive and time-consuming, requiring substantial training and expertise due to fleeting skills. These problems are magnified in nonendemic regions where light microscopy to diagnose malaria is infrequently performed, resulting in missed diagnosis, misidentification of Plasmodium species, and therapeutic delays. Methods using advances in technology have been evaluated as alternatives to light microscopy. While these methods have varying strengths and weaknesses, they are limited by equipment, supplies, expertise, cost, time, applicability in acute infection, and/or availability. New immunochromatographic rapid diagnostic tests (RDTs) for malaria were introduced. Table 2. Comparison of methods for diagnosing Malaria in blood Parameter Sensitivity (parasites/ul) Microscopy PCR Fluorescence RDT HRP-II RDT pldh 50 5 50 >100 >100 Specificity All species All species P.falciparum good, others difficult P.falciparum only P.falciparum, P.vivax, P.ovale and P.malariae only with pldh Parasite density or parasitemuia Yes No 50 >100 >100 Time for result 30~60 min 24 h 30~60 min 15~30 min 15~30 min Skill Level High High Moderate Low Low Equipment Microscopy PCR apparatus Fluorescence microscopy None None Cost/test Low High Moderate Moderate Moderate

Histidine Rich Protein-II (HRP-II) The antigen is expressed only by P.falciparum trophozoites. The antigen can be detected in erythrocytes, serum, plasma, cerebrospinal fluid and even urine as a secreted water-soluble protein. It generally takes around two weeks after successful treatment for HRP-II-based tests to turn negative, but may take as long as one month, which compromises their value in the detection of active infection. Since HPR-II is expressed only by P. falciparum, these tests will give negative results with samples containing only P. vivax, P. ovale, or P. malariae; many cases of non-falciparum malaria may therefore be misdiagnosed as malaria negative (some P.falciparum strains also don t have HRP-II) pldh panldh P. falciparum lactate dehydrogenase (pldh) is a 33 kda oxidoreductase. It is the last enzyme of the glycolytic pathway, essential for ATP generation and one of the most abundant enzymes expressed by P. falciparum. pldh is similar to pgludh. LDH from P. vivax, P. malariae, and P. ovale exhibit 90~92% identity to pldh from P. falciparum. Table 3. Characteristics of HRP-II and pldh antigen Description Sensitivity HRP-II Approx. >99.9% (100 parasite/ul) pldh Approx. >95% (100 parasite/ul) Specificity Approx. >99.5% Approx. >99.5% False Result False positive result for 7~14 days following chemotherapy False negative result in low levels of parasites False positive result for gametocytes Differential Detection P.f only P.f and other species (Pan) Detection period Purpose Human life cycle except matured gametocytes Suitable test for prevalence of P.f All stage of human life cycle including gametocyte For monitoring with therapy Suitable test for prevalence of P. vivax

Humasis Malaria Antigen test can offer significant benefits in malaria management as followings: A clear benefit will occur in health outcomes Allow more rational use of anti-malarial drugs The accuracy of Humasis Malaria Antigen test is quality controlled Protected from high temperature Affordable Price Potential uses for Humasis Malaria Antigen test Outbreak investigation and malaria prevalence surveys. Diagnosis by health workers distant from good microscopy services. After-hours diagnosis in hospital or clinics. Remote diagnosis in organized workforces in endemic area.

Test Methods 4. Clean the fingertip with an alcohol pad and let dry. 5. Take a lancet and make a quick deep stab on the side of the finger. 6. Take 5uL of whole blood by a loop. In order to collect the correct amount of blood, lay the loop and fill a film of blood completely across the loop. Whole blood 1. FIRST, read carefully the instructions on how to use Humasis Malaria Antigen test kit. 2. Open the package and look for the following. 1) Test device individually packaged with 2) Buffer a foil-pouch and with a silica gel in it. 7. Drop the specimen in a specimen insertion-hole. 3) Disposable sample loop(5ul) 4) Lancet 5) Alcohol prep pad Open the foil pouch, and look for the following. 8. Add 4 drops of buffer(approximately 120uL). 3. Next, look at the expiry date at the back of the foil pouch. Use anothe tet device if the expiry date has passed. 9. Wait for 30 minutes and then read the result. Do not interpret the test result after 30 minutes.

Interpretation of the results Malaria P.f/Pan Antigen Test Negative Positive 1. Positive for P.f 2. Positive for P.v or P.o or P.m 3. Positive for P.f and P.v or P.o or P.m The colored band in P.f region can be appeared after the medical treatment. Invalid Malaria P.f/P.v Antigen Test Negative Positive 1. Positive for P.f 2. Positive for P.v Invalid

Specification Specification Humasis Malaria P.f/Pan Antigen Test Humasis Malaria P.f/P.v Antigen Test Humasis Malaria P.f Antigen Test Test Method Lateral flow test Result within Specimen Semple volume Target antigen HRP-II for P.f and pldh for P.f, P.v, P.o and P.m 15-30minutes Whole blood or finger punctured blood 5uL HRP-II for P.f and pldh for P.v HRP-II for P.f Sensitivity P.f: 99.9% P.pan: 95.6% P.f: 99.9% P.v: 95.6% 99.9% Specificity Stability Storage 99.5% 99.5% 99.5% 24 months 1~30 C Contents Humasis Malaria Antigen Test 25 Devices Disposable sample dispensing loop (5uL) 25 ea Dilution buffer 1 bottle Alcohol prep pad 25 ea Lancet 25 ea Instruction Manual 1 ea

Ordering Information Test Device HUMASIS Malaria P.f/Pan Antigen Test HUMASIS Malaria P.f/P.v Antigen Test HUMASIS Malaria P.f Antigen Test Cat. No. AMAL-7025 AMFV-7025 AMPF-7025 Contact information HUMASIS CO., LTD. Rm. 504 SHINWON VISION TOWER 898 Hogye-dong, Dongan-gu, Anyang-si, Gyeonggi-do, 431-836, KOREA Tel: 031-478-8591 Fax: 031-478-8586 Purchase Product: +82-31-478-8591, e-mail: question@humasis.com Technical Service: +82-31-478-8595, e-mail: customerservice@humasis.com For more information, please visit our website www.humasis.com TR2106. Rev.01 SEP 2011